Inhibition of Interaction of PSD93 and PSD95 with nNOS and NMDA Receptors

ABSTRACT

PSD-95/SAP90 antisense-treated animals not only experience a significant decrease in MAC for isoflurane, but also experience an attenuation in the NMDA-induced increase in isoflurane MAC. PSD-95/SAP90 appears to mediate the role of the NMDA receptor in determining the MAC of inhalational anesthetics. Suppression of the expression of PSD-95/SAP90 in the spinal cord significantly attenuates responses to painful stimuli mediated through the N-methyl-D-aspartate receptor activation. In spinal cord neurons PSD-95/SAP90 interacts with the N-methyl-D-aspartate receptor subunits 2A/2B. Activation of the N-methyl-D-aspartate receptor in spinal hyperalgesia results in association of the N-methyl-D-aspartate receptor with PSD-95/SAP90. PSD-95/SAP90 is required for hyperalgesia triggered via the N-methyl-D-aspartate receptor at the spinal cord level.

This application claims the benefit of provisional application Ser. No.60/242,580 filed October 23, 2000, and Ser. No. 60/203,894 filed May 12,2000, the entire contents of which are expressly incorporated herein.

This invention was made using funds from the U.S. government undergrants from the National Institutes of Health numbered RO GM49111 andRO1 HL39706. The U.S. government therefore retains certain rights in theinvention.

BACKGROUND OF THE INVENTION

The potency of anesthetic agents to inhibit the ability of a patient torespond with movement to painful stimuli has long been used as a test ofanesthetic action. This potency, characterized by its ED₅₀, is widelyknown as the minimum alveolar concentration (MAC). Several lines ofevidence have shown that spinal NMDA receptor activation might play akey role in the processing of nociceptive information^(1,29-30) and inthe determination of the MAC of inhalational anesthetics.³¹⁻³³ Forexample, the NMDA receptors are distributed mainly in the superficiallaminae of the spinal cord.^(12,28) Both repetitive C-fiber stimulationand direct application of glutamate or NMDA produce spinal neuronalsensitization and enhance responsiveness, which can be blocked by NMDAreceptor antagonists.^(1,34-36) Behavioral studies demonstrate thatspinal administration of NMDA produces thermal hyperalgesia, caudallydirected scratching and biting, and exaggerated responsiveness to lighttouch.^(8,37-39) Moreover, antagonism of the spinal NMDA receptorsproduces antinociception in numerous animal models of pain³⁹⁻⁴⁴ andreduction in the MAC of isoflurane.³¹⁻³³ However, the molecularmechanisms underlying these actions remain unknown.

The postsynaptic density (PSD), a highly organized cytoskeletalstructure found adjacent to the postsynaptic membrane of excitatorysynapses, is believed to play a role in the organization of receptorsand related proteins involved in synaptic signaling.⁴⁵⁻⁵⁵ A number ofproteins enriched in the PSD have been characterized.⁴⁷⁻⁴⁸ One of theseproteins, postsynaptic density-95 (PSD-95)/synapse-associated protein-90(SAP90), is an abundant scaffolding molecule that binds and clusters theNMDA receptor preferentially at synapses in the brain and spinalcord.^(3,4,5,7,9,49) This raises the possibility that PSD-95/SAP90 mightbe involved in many physiological and pathophysiologic actions triggeredvia the NMDA and perhaps other receptors in the central nervous system.Indeed, suppression of PSD-95/SAP90 expression attenuated excitotoxicityproduced via NMDA receptor activity in brain neurons.²³ The lack ofPSD-95/SAP90 revealed an enhanced NMDA-dependent long-term potentiationand impaired learning.¹⁶

The role of the N-methyl-D-aspartate (NMDA) receptor in spinalhyperalgesia has been demonstrated by behavioral, electrophysiologicaland neurochemical findings.^(1,8,21,26) However, the molecularmechanisms underlying these actions are unclear. The NMDA receptorconsists of two distinct types of subunits: NMDAR1 (NR1) and NMDAR2A-D(NR2A-D).¹⁹ The C-termini of the NR2 subunits interact with PSD-95/SAP90and other members of the membrane-associated guanylate kinase (MAGUK)family in the brain.^(2,6,9,10,17,20) This raises the possibility thatthe sensory hyperalgesia produced through NMDA receptor activation isdetermined by NMDA receptor-bound proteins of the MAGUK family in thespinal cord.

There is a need in the art for new ways of treating and preventinghyperalgesia and chronic and acute pain. In addition, there is a need inthe art for new and safer ways of rendering patients unconscious viageneral anesthesia or by sedating them.

SUMMARY OF THE INVENTION

One embodiment of the invention provides a method for relieving acute orchronic pain. According to the method an effective amount of an agentwhich inhibits expression of PSD93 or PSD95 is administered to a subjectin need of pain relief. The agent relieves acute or chronic painexperienced by the subject.

Another embodiment of the invention provides a method for treating orpreventing hyperalgesia. According to the method an effective amount ofan agent which inhibits expression of PSD93 or PSD95 is administered toa subject who has or is at risk of developing hyperalgesia. Theadministration relieves or prevents hyperalgesia experienced by thesubject.

Another aspect of the invention is a method of reducing a threshold foranesthesia. An anesthetic and an agent which inhibits expression ofPSD93 or PSD95 are administered to a subject. The amount of anestheticadministered achieves a desired anesthetic effect even though the amountadministered is less than the amount required in the absence of theagent to achieve the desired anesthetic effect. This minimizes theserious side effects of the anesthetics including cardiovascular andrespiratory depression.

The present invention also provides an isolated and purified antisensepolynucleotide which is complementary to PSD95 or PSD93 mRNA.

Another embodiment of the invention is a method for relieving acute orchronic pain. An effective amount of an agent which inhibits interactionof a first protein selected from the group consisting of PSD93 andPSD95, with a second protein selected from the group consisting of nNOSand NMDA receptor, is administered to a subject in need thereof. Theagent does not cause cardiovascular or respiratory depression. Theadministration relieves acute or chronic pain experienced by thesubject.

Also provided is an alternative method for treating or preventinghyperalgesia. An effective amount of an agent which inhibits interactionof a first protein selected from the group consisting of PSD93 andPSD95, with a second protein selected from the group consisting of nNOSand NMDA receptor, is administered to a patient experiencinghyperalgesia or who is at risk of developing hyperalgesia. The agentdoes not cause cardiovascular or respiratory depression. Hyperalgesiaexperienced by the subject is relieved or prevented by theadministration.

Also provided by the present invention is a method of reducing athreshold for anesthesia. An anesthetic and an agent which inhibitsinteraction of a first protein selected from the group consisting ofPSD93 and PSD95, with a second protein selected from the groupconsisting of nNOS and NMDA receptor, are administered to a subject. Theagent does not cause cardiovascular or respiratory depression. Theamount of anesthetic administered is less than the amount required inthe absence of the agent to achieve a desired anesthetic effect. Thedesired anesthetic effect is thus achieved.

The present invention also provides a method of anesthetizing a subject.An agent which inhibits expression of PSD93 or PSD95 is administered toa subject. The agent renders the subject unconscious or sedated.

Another embodiment of the invention provides a method of anesthetizingor sedating a subject. An agent which inhibits interaction of a firstprotein selected from the group consisting of PSD93 and PSD95, with asecond protein selected from the group consisting of nNOS and NMDAreceptor, is administered to a patient. The agent does not causecardiovascular or respiratory depression. The agent renders the subjectunconscious or sedated.

Yet another aspect of the invention is a method of screening forsubstances useful for relieving pain or inducing unconsciousness orsedation. A test substance is contacted with a first protein and asecond protein under conditions where the first protein and the secondprotein bind to each other. The first protein is selected from the groupconsisting of PSD93, PSD95, and a combination thereof. The secondprotein is selected from the group consisting of nNOS, NMDA receptor,NR2A subunit, NR2B subunit, and combinations thereof. The mixture ofproteins is assayed to determine the binding of the first protein to thesecond protein. Any parameter which reflects that binding can beassayed. Such parameters include the amount of free nNOS, the amount offree PSD93, the amount of free PSD95, the amount of free NMDA receptor,the amount of free NR2A subunit, the amount of free NR2B subunit, theamount of bound nNOS, the amount of bound PSD93, the amount of boundPSD95, the amount of bound NMDA receptor, the amount of bound NR2Asubunit, the amount of bound NR2B subunit and combinations of them. Atest substance which increases the amount of free nNOS, free PSD93, freePSD95, free NMDA receptor, free NR2A subunit, or free NR2B subunit, orwhich decreases the amount of bound nNOS, bound PSD93, bound PSD95,bound NMDA receptor, bound NR2A subunit, or bound NR2B subunit isidentified as a candidate drug for relieving pain or inducingunconsciousness or sedation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Expression of PSD-95/SAP90 mRNA and protein in thespinal cord. In FIG. 1Aa, immunoblot showing the expression ofPSD-95/SAP90 in the PSD fractions of the spinal cord (SC), dorsal rootganglion (DRG) and other brain regions (as positive controls) in thenormal rats. HI: hippocampus; CO: cortex; CE: cerebellum. In FIG. 1Ab,immunoblot showing representative effects of PSD-95/SAP90 antisense(AS), missense (MS) and sense (SE) ONDs, as well as saline (SA), on theexpression of PSD-95/SAP90, nNOS and NR2A/2B in the spinal cord. PC:positive control tissue from hippocampus. Asterisk: non-specific band bythe secondary antibody, useful to control for protein loading and blotexposure times. In FIG. 1B, RT-PCR analysis showed that 0.737 Kb mRNAwas detected in the spinal cord and other brain regions (hippocampus,cortex, cerebellum and brainstem), but not in muscle. PCR product wasdirectly cloned into the TA cloning vector and verified as PSD-95/SAP90by automatic DNA sequencing. β-actin mRNA was used as a loading control.

FIGS. 2A and 2B. Distribution of PSD-95/SAP90 immunoreactivity in lumbarenlargement segments of the spinal cord. The PSD-95/SAP90immunoreactivity was localized mainly in lamina I and outer lamina II(A). Under high magnification, many PSD-95/SAP90 immunoreactive punctawere observed (B). Scale bars: 200 μm in A; 40 μm in B.

FIG. 3. Identification of a ternary complex assembled by PSD-95/SAP90with NR2A/2B and nNOS in the spinal cord neurons. PSD-95/SAP90 antibodyimmunoprecipitated not only PSD-95/SAP90 but also NR2A/2B and nNOS. Incontrast, endothelial NOS (eNOS) was not immunoprecipitated byPSD-95/SAP90 antibody. Ten μg protein was loaded in INPUT lane and 100μg in other lanes.

FIG. 4. Effect of intrathecal administration of NMDA on isoflurane MACin the saline- and PSD-95/SAP90 antisense ODN-treated groups. Data arepresented as mean±SD. n=5 animals for each group, except n=14 for thesaline-treated (control) group. ** Significantly different from control(P<0.01).

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present inventors that PSD95 and PSD93 mediatethe interaction of NMDA receptors and nNOS in the spinal cord, and areinvolved in generating responses to painful stimuli. The inventors havefound that inhibition of the interaction of NMDA receptors and nNOS viaPSD95 and PSD93 can attenuate responses to painful stimuli, as well aslower thresholds for anesthetics. Moreover, we have found thatinhalational anesthetics themselves inhibit the interaction of NMDAreceptors and nNOS via PSD95 and PSD93. Thus new and improvedanesthetics and sedatives can be identified using the identifiedinteraction as an assay system.

Acute or chronic pain can be relieved or prevented according to thepresent invention by administering to a subject an effective amount ofan agent which inhibits expression of PSD93 or PSD95. The agents of thepresent invention also can be used to treat or prevent hyperalgesia, aswell as to reduce a threshold for anesthesia. The agent used can be anantisense oligonucleotide (ODN) which is complementary to mRNA encodingPSD93 or PSD95. Preferably the antisense oligonucleotide iscomplementary to nucleotides encoding a PDZ domain. More preferably theantisense oligonucleotide is complementary to nucleotides 241 to 258 ofPSD95. Any agent which acts to specifically inhibit transcription ortranslation of PSD93 or PSD95 can be used. Oligonucleotides useful inthe invention can be naked oligonucleotides or can be administered in avector, liposome, particle or other protective formulation. If in avector, the vector can express RNA molecules which are complementary tothe native PSD93 or PSD95 mRNAs. Also encompassed by the presentinvention are oligonucleotides which contain nucleotide analoguemoieties to render the oligonucleotides less susceptible to enzymaticdegradation. Suitable nucleotide analogue moieties are known in the artand include phosphorothioates.

Agents according to the present invention can be administered any wayknown in the art which is convenient and efficient for the particularagent and the application. Preferably the agent is administeredintrathecally, per os, or intravenously. However, other means can beused as appropriate, including subdermal, subcutaneous, rectal,intraperitoneal, subarachnoid, caudal, epidural, inhalational, andintramuscular administrations. Anesthetics and sedatives used in themethods of the present invention can also be administered by any ofthese same means. Preferred anesthetics according to the invention areinhalational anesthetics, including halothane, isoflurane, desflurane,xenon, and sevoflurane.

Compositions are provided for inhibiting expression of PSD95 or PSD93.Such compositions comprises an isolated and purified antisensepolynucleotide which is complementary to PSD95 or PSD93 mRNA. Preferablythe polynucleotide is complementary to nucleotides encoding a PDZdomain. Any of the three such domains can be targeted, although thethird such domain, i.e., the C-terminal PDZ domain, may be the mosteffective. One particular oligonucleotide which has been found to beeffective is complementary to nucleotides 241 to 258 of PSD95. Theanalogous nucleotides of PSD93 can also be used. The polynucleotide canbe formulated in a pharmaceutically acceptable vehicle so that it can beused to prevent pain or to lower an anesthetic or sedative threshold.Particular vehicles which are suitable for intrathecal or inhalationaltherapy can be advantageously used. The formulations can be in liquid orvapor form. They can be vaporized by bubbling a gas through them.Preferably the formulations of the invention will be manufactured underregulatory-approved conditions for administration to humans.Requirements for such formulations typically include sterility andfreedom from pyrogens.

Not only can agents which specifically inhibit the expression of PSD93or PSD95 be used in the methods of the present invention, but alsoagents which inhibit the interaction of PDS93 or PSD95 with either nNOSor NMDA receptors. Such agents can be used for the same purposes asdiscussed above, for relieving acute or chronic pain, for reducing thethreshold for anesthetics and sedatives, and for anesthetizing andsedating patients directly. Agents useful according to the presentinvention do not cause cardiovascular or respiratory depression. Suchagents can be administered to the same populations of patients asdiscussed above, i.e., those in need of anesthesia, those in need ofrelief from chronic or acute pain, and those who experience hyperalgesiaor are at risk of developing hyperalgesia. Such patients include thosewhose pain is mechanical, thermal, neuropathic, or inflammatory inorigin.

Protein interaction-inhibitory agents of the invention preferably bindto a PDZ domain of any of the binding participants, including nNOS, NMDAreceptors, PDS93 or PDS95. Typically and preferably the agent does notimpair motor function, i.e., locomotion. Such agents can be identifiedby any of a number of screening techniques which rely on the inhibitionof expression or interactions of PDS93 or PDS95. Generally, testsubstances are contacted with a first protein and a second protein underconditions where the first protein and the second protein bind to eachother. The first protein is PSD93, PSD95, or a combination the twoproteins. The second protein can be nNOS, NMDA receptor, NR2A subunit,NR2B subunit, or combinations of these proteins. Fusion proteins whichcontain all or relevant binding portions of these proteins can be used,as is desirable for ease of detectability or purification and handling.The amount of protein which is bound or free in the presence and absenceof the test substance can be determined by any techniques known in theart. Test substances which increase the amount of free binding partnersor which decrease the amount of bound binding partners are identified ascandidate drugs for relieving pain or inducing unconsciousness orsedation.

Many protein-protein binding assays are known in the art and any suchformat or technique can be used as is convenient. In some assays theproteins are contacted in vitro. In other assays the proteins are inyeast cells containing recombinant forms of the first and secondproteins, and the test substance is contacted with the whole yeastcells. Such assays include the well-known two hybrid assays, in whichbinding of two binding partners reconstitutes a transcriptionalactivating activity. In these assays the first and second bindingpartners are each fused to a first and second yeast protein whichreconstitute a functional transcriptional activator when brought intophysical proximity by binding of the first recombinant protein to thesecond recombinant protein. Colorimetric, enzymatic, or growth assayscan be used to determine the transcriptional activation reconstitution.Candidates which are identified as having inhibitory activity in suchassays can be further tested in an animal to determine if the candidatedrug relieves pain or induces unconsciousness or sedation.

PSD-95/SAP90 antisense-treated animals not only experience a significantdecrease in MAC for isoflurane, but also experience an attenuation inthe NMDA-induced increase in isoflurane MAC. PSD-95/SAP90 appears tomediate the role of the NMDA receptor in determining the MAC ofinhalational anesthetics. Suppression of the expression of PSD-95/SAP90in the spinal cord significantly attenuates responses to painful stimulimediated through the N-methyl-D-aspartate receptor activation. In spinalcord neurons PSD-95/SAP90 interacts with the N-methyl-D-aspartatereceptor subunits 2A/2B. Activation of the N-methyl-D-aspartate receptorin spinal hyperalgesia results in association of theN-methyl-D-aspartate receptor with PSD-95/SAP90. PSD-95/SAP90 isrequired for hyperalgesia triggered via the N-methyl-D-aspartatereceptor at the spinal cord level.

The pretreatment of PSD-95/SAP90 antisense ODN but not sense or missenseODN produced a remarkable reduction in isoflurane MAC. This was notaccompanied by changes in ether blood pressure or heart rate.Furthermore, the PSD-95/SAP90 antisense ODN blocked NMDA-inducedincrease in isoflurane MAC. The deficiency of PSD-95/SAP90 expressionmay produce anesthetic and analgesic actions at the spinal cord leveland PSD-95/SAP90 might mediate the role of the NMDA receptor indetermining the MAC of inhalational anesthetics.

Antisense ODNs have been widely used as research tools, and even asdrugs in clinical trials. Antisense ODNs inhibit protein expression bythe mechanisms of (1) steric blockade of ribosomal subunit attachment tomRNA at the 5′ cap site; (2) interference with proper mRNA splicingthrough antisense binding to splice donor or splice acceptor sites; (3)RNase-H-mediated degradation of hybridized mRNA.¹⁸ The proper design andcontrols of experiments are critical in demonstrating a true antisenseeffect. The specificity of intrathecal treatment with PSD-95/SAP90antisense ODN has been shown. First, we designed the standard controlsof equivalent sense sequence and missense ODNs. Neither had any effecton the isoflurane MAC. This indicates the specificity of the inhibitionobserved with the antisense ODN. Second, all of the ODNs had beensearched to exclude non-specificity of the sense or antisense ODNs andto show that missense ODN did not match any confounding sequences in theGenBank database. Moreover, our previous results have demonstrated thatantisense ODNs only suppressed the expression of PSD-95/SAP90 but notthe expression of NMDA receptor subunits NR2A/2B, neuronal nitric oxidesynthase or SAP102 (a protein that is closely related to the targetedprotein) in the spinal cord.⁴⁹ The effects observed following treatmentwith the PSD-95/SAP90 antisense ODN are unlikely to be explained bychanges in the expression of other proteins. Finally, the antisense ODNsat the doses used only affected isoflurane MAC without untoward effectsin any of the treated animals including the antisense groups.Considering these several lines of evidence, we believe that the effectswe have described may be due to a direct and selective interference ofthe antisense ODN with mRNA transcripts of PSD-95/SAP90 and to theblockade of protein production via binding to the nucleotides ofPSD-95/SAP90 mRNA.

The regional expression and function of PSD-95/SAP90 in the mammalianbrain have been investigated using a variety of experimentalapproaches.^(3,4,5,7,9) PSD-95/SAP90 immunoreactivity was found mainlyin cortex, hippocampus and cerebellum.⁵⁴⁻²⁶ In brain neurons,suppression of PSD-95/SAP90 expression that selectively disruptedphysical linkage of the NMDA receptor with neuronal nitric oxidesynthase has been demonstrated to attenuate excitotoxicity andCa2+-activated nitric oxide production via NMDA receptor activity.²³Mice carrying a targeted mutation in the PSD-95/SAP90 gene showed anenhanced NMDA-dependent long-term potentiation and impaired learning.¹⁶Recently, we found that PSD-95/SAP90's mRNA and protein also wereenriched in the spinal cord and selectively distributed in thesuperficial dorsal horn, where PSD-95/SAP90 expression overlapped withthat of the NMDA receptor.¹²⁻²⁸ In the spinal neurons, PSD-95/SAP90interacted with the NMDA receptor subunits 2A/2B.⁴⁹ Behavioral studiesshowed that intrathecal administration of antisense ODN for PSD-95/SAP90significantly attenuated facilitation of the tail-flick reflex triggeredthrough the NMDA receptor activation.⁴⁹ The evidence above indicatesthat activation of the NMDA receptor in spinal hyperalgesia results inassociation of the NMDA receptor with PSD-95/SAP90 and that PSD-95/SAP90is required for the spinal mechanisms of hyperalgesia. This suggeststhat PSD-95/SAP90 may be involved in the processing of pain and thatdeficiency of PSD-95/SAP90 may produce analgesic action at the spinalcord level. Such an action is consistent with the effect of thedeficiency of PSD-95/SAP90 on MAC. Doses of antisense ODNs did not causemotor and general behavioral dysfunction when administered intrathecallyin rats. The effect of suppression of spinal PSD-95/SAP90 expressionthat resulted in the reduction in MAC may be due to effects on analgesiaalone. However, PSD-95/SAP90 has been demonstrated to be involved in themechanisms of long-term potentiation and learning.¹⁶ An effect ofantisense ODN on righting reflex was not observed. The possibility ofthese actions of the antisense ODNs in the central nervous system couldnot be ruled out from the current study since the intrathecal antisenseeffect had a segmental nature.

A role for the NMDA receptors in determining the MAC of inhalationalanesthetics is suggested by the fact that the systemic or intrathecaladministration of NMDA antagonists significantly reduces the MAC ofisoflurane in rats, which is completely reversed to control level byintrathecal administration of NMDA.³¹⁻³³ The current study furtherindicated that intrathecal administration of NMDA increased the MAC ofisoflurane in saline-treated rats. Interestingly, in antisenseODN-treated rats, intrathecal injection of NMDA did not affect the MACof isoflurane. PSD-95/SAP90 localization completely overlapped with theNMDA receptor subunits 2A/2B in spinal superficial dorsal horn.⁴⁹Furthermore, the PSD-95/SAP90 antibody was able to immunoprecipitate notonly PSD-95/SAP90 but also NR2A/2B in vivo.⁴⁹ These findings demonstratethat PSD-95/SAP90 interacts with NR2A/2B in the spinal cord in vivo.Combined with the current results, it is suggested that PSD-95/SAP90 isessential for the actions of the NMDA receptor in determining the MAC ofinhalational anesthetics.

In our experiments, no significant hemodynamic effects were observed inthe ODN-treated animals during isoflurane anesthesia. However,intrathecal administration of NMDA resulted in a significant increase insystolic and diastolic blood pressure during isoflurane anesthesia inboth the saline- and antisense ODN-treated groups. It has beendemonstrated that sympathetic preganglionic neurons located in theintermediate nucleus of the spinal cord are integral elements in theneural pathway linking the central nervous system to sympathetic nervessupplying the heart and blood vessels.^(57,58) The effects of NMDA onblood pressure may be due to the involvement of the NMDA receptor inregulation of sympathetic output at the spinal cord level. Inimmunohistochemical studies, glutamate and its receptors were found inthe intermediolateral nucleus of the thoracic spinal cord.^(59,60)Intrathecal administration of NMDA at the T10 level increased arterialpressure. This action was blocked by NMDA receptor antagonists. ^(61,62)It is likely that NMDA, administered intrathecally at the lumbar level,activates spinal sympathetic activity in the intermediolateral nucleusand produces the increase in blood pressure. The antisense ODNs had noeffect on the hemodynamics or on the NMDA-induced increase in bloodpressure, a finding which is consistent with our previous observationthat PSD-95/SAP90 was absent or present at extremely low levels in theintermediolateral nucleus of the spinal cord.⁴⁹ It could be that thesecond message signaling pathways in the somatic and the sympatheticnervous systems are different with respect to the NMDA receptor. Hong etal⁶¹ and West et al⁶² reported that microinjection of NMDA into theintermediolateral nucleus at the spinal T₂ level or intrathecalinjection of NMDA at the T₁₀ level produced an increase in heart rate.Interestingly, the effect of NMDA on heart rate was not observed ineither saline- or antisense ODN-treated groups in the present study. Thereason for this discrepancy between the previous and the present studiesis not clear and may be due to a difference in anesthetic agents(isoflurane in the present study vs urethane, chloral hydrate and sodiumpentobarbitone). It is interesting to note that intrathecaladministration of the NMDA receptor antagonist, APV, produced adose-related decrease in arterial pressure but not in heartrate.^(61,63) These data suggest that there is a tonic activation of theNMDA receptor in the spinal sympathetic pathway to the vessels but notto the heart.

MAC for isoflurane was significantly decreased and the NMDA-inducedincrease in isoflurane MAC was attenuated in the PSD-95/SAP90antisense-treated animals. The binding of PSD-95/SAP90 to the NMDAreceptor preferentially at synapses in the spinal cord and brainsuggests that PSD-95/SAP90 may mediate the role of the NMDA receptor indetermining the MAC of inhalational anesthetics.

EXAMPLES Example 1

This example demonstrates that PSD-95 is necessary for thermalhyperalgesia.

To examine whether PSD-95/SAP90 was required for thermal hyperalgesiatriggered through NMDA receptor activation, we made an antisenseoligonucletide (OND) corresponding to the PDZ domain nucleotides 241 to258 (5′-TGTGATCTCCTCATACTC-3′; SEQ ID NO: 1) of rat PSD-95/SAP90 mRNA,as well as the sense OND and missense OND (5′-AAGCCCTTGTTCCCATTT-3′; SEQID NO: 2). All of the ONDs were compared to the Gene Bank database(GenBank accession number M96853) and found not to be complementary toany registered nucleotide sequences. The effects of antisense, sense andmissense ONDs on baseline and NMDA-induced tail-flick latencies wereassessed. Consistent with previous studies, ^(14,15,24,26) intrathecaladministration of NMDA at 5 nM/10 μl (n=6) (data not shown) or 10 nM/10μl (n=12) induced a facilitation of the tail-flick reflex (The baselinetail-flick latency was reduced from 6.58±0.57 to 4.88±0.41 seconds.p<0.01) (Table 1). We found that the NMDA-produced facilitation of thetail-flick reflex was attenuated in rats pretreated with antisense ONDs(25 μg/10 μl and 50 μg/10 μl every 24 h for 4 days; n=6 each group) butnot in those pretreated with sense OND (50 μg/10 μl every 24 h for 4days; n=6) or missense OND (50 μg/10 μl every 24 h for 4 days; n=6)(Table 1). Antisense OND given intrathecally at 25 and 50 μgdramatically prevented the NMDA-induced decrease of the tail-flicklatency by 55% (p<0.05) and 82% (p<0.01), respectively. When these ratstreated with antisense OND were allowed to recover for an additionalfour days, their tail flick latency in response to NMDA stimulationreturned to normal. To identify that NMDA-induced thermal hyperalgesiawas produced specifically through NMDA receptor activation but notnon-NMDA receptor activation, we observed the effects of a selectiveNMDA receptor antagonist, MK-801, and a selective non-NMDA receptorantagonist, DNQX, on NMDA-induced facilitation of the tail-flick reflex.As shown in Table 1, intrathecal MK-801 at 10 nM/10 μl (n=6) completelyabolished facilitation of the tail-flick reflex stimulated by NMDA(p<0.01), while intrathecal DNQX at 20 nM/101 (n=6) had no effect(p>0.05). The baseline thermal reflex is generally considered to bemediated via non-NMDA receptor mechanisms.^(13-15,26) Antisense OND forPSD-95/SAP90 did not affect baseline tail-flick latency (percentagechange of TF latency was 0.44±1.95) compared to the control group; Nordid sense and missense ONDs (percentage changes of TF latencies were−0.82±1.94 and −2.05±1.57, respectively). In addition, motor weakness ordysfunction was not observed in locomotor tests (including placingreflex, grasping reflex and righting reflex) in any of the treatedanimals including the antisense groups (data not shown).

Example 2

This example shows that PSD-95 antisense oligonucleotide actsspecifically to inhibit PSD-95 expression.

Antisense ONDs, widely used as research tools and even as drugs inclinical trials, inhibit protein expression by the mechanisms of (1)steric blockade of ribosomal subunit attachment to mRNA at the 5′ capsite; (2) interference with proper mRNA splicing through antisensebinding to splice donor or splice acceptor sites; (3) Rnase-H-mediateddegradation of hybridized mRNA.¹⁸ To further examine whether the actionof antisense OND for PSD-95/SAP90 above was specifically due toselective decrease or lack of PSD-95/SAP90 but not other proteins in thespinal cord, we detected PSD-95/SAP90, NMDA receptor subunits 2A/2B(NR2A/2B), neuronal nitric oxide synthase (nNOS) and SAP-102 inhomogenates from crude lumbar enlargement segments in the normal,saline-treated (control) and OND-treated rats. PSD-95/SAP90 protein wasenriched in the postsynaptic density (PSD) fraction of the spinal cordin normal, control, sense OND- and missense OND-treated groups (FIG. 1Aa and b). In contrast, in the antisense OND-treated group, PSD-95/SAP90expression was suppressed to <15% of control (FIG. 1Ab). No significantchange in expression of NR2A/2B, nNOS and SAP-102 was found in normal,control or OND-treated animals (FIG. 1Ab). It is likely that theantisense OND for PSD-95/SAP90 selectively interferes with mRNAtranscription of PSD-95/SAP90 and blocks production of the protein viabinding to the nucleotides of PSD-95/SAP90 mRNA. Combined with thebehavioral results above, it is suggested that the expression ofPSD-95/SAP90 in the spinal cord might be critical for spinal thermalhyperalgesia via NMDA receptor activation.

Example 3

This example demonstrates the expression and localization of PSD95 inthe spinal cord, as well as the colocalization with NMDA receptors andnNOS.

To provide further support for the role of PSD-95/SAP90 in spinalhyperalgesia, we examined the expression of PSD-95/SAP90 and theinteraction of PSD-95/SAP90 with the NMDA receptor in the spinal cord.The regional expression and function of PSD-95/SAP90 in the mammalianbrain have been investigated using a variety of experimentalapproaches.^(3-5,7,9,16) To our knowledge, however, there are noprevious reports of its expression or function in adult spinal cord.Thus, RNA for messages encoding the PSD-95/SAP90 protein was extractedfrom tissues of the spinal cord, other regions of the brain (as positivecontrols), and muscle (as a negative control). This RNA was probed withthe use of RT-PCR analysis. A 0.735 Kb mRNA was detected in the spinalcord and regions of brain (hippocampus, cortex, cerebellum andbrainstem), but not in muscle (FIG. 1B). The PCR product then wasdirectly cloned into the TA cloning vector and verified as PSD-95/SAP90by automatic DNA sequencing. Furthermore, the distribution ofPSD-95/SAP90 immunoreactivity in the spinal cord was observed. Asillustrated in FIG. 2A, PSD-95/SAP90 immunoreactivity was found in thespinal cord and distributed mainly in lamina I and outer lamina II.Under high magnification, many PSD-95/SAP90 immunoreactive puncta wereobserved (FIG. 2B). The superficial dorsal horn not only contains manyinterneurons and their processes but also receives the processes fromthe deep dorsal horn neurons, the primary afferent termini from theperiphery and the descending fibers from supraspinal structures.²² SincePSD-95/SAP90 is specifically localized at synapses and has been foundboth pre- and post-synaptically in the brain,^(3,4) we investigated thesources of PSD-95/SAP90 immunoreactive puncta in the superficial dorsalhorn. In the dorsal root ganglion of normal rat, no PSD-95/SAP90 proteinwas detected (FIG. 1Aa). Also, there was no change in the density ofPSD-95/SAP90 immunoreactivity in the superficial dorsal horn afterunilateral spinal nerve cut or bilateral dorsolateral fasciculi cut(data not shown). More importantly, PSD-95/SAP90 mRNA was detected andPSD-95/SAP90 expression from antisense OND-treated rats wassignificantly suppressed in the spinal cord as described above. Thesedata indicate that PSD-95/SAP90 in the superficial dorsal horn, to agreat extent, is intrinsic to the spinal cord. The superficial dorsalhorn is the primary center for processing noxious stimulation.²² Thearea-specific expression and distribution of PSD-95/SAP90 in the spinalcord suggest that PSD-95/SAP90 has important implications for themechanisms of nociceptive processing at the spinal cord level. The NMDAreceptor has been demonstrated to mainly locate in lamina I and outerlamina II of the spinal cord.^(12,28) Combined with the present data,the NMDA receptor completely overlapped with PSD-95/SAP90 in the spinaldorsal horn. It is suggested that PSD-95/SAP90 may co-localize andinteract with the NMDA receptor in the spinal cord neurons. This wasfurther confirmed with the use of co-immunoprecipitation, demonstratingthat the PSD-95/SAP90 antibody was able to immunoprecipitate not onlyPSD-95/SAP90 but also NR2A/2B and nNOS in vivo (FIG. 3). In contrast,endothelial NOS (eNOS) was not immunoprecipitated with the PSD-95/SAP90antibody (FIG. 3). These findings show that PSD-95/SAP90 interacts withNR2A/2B in the spinal cord in vivo, suggesting that glutamatestimulation of the NMDA receptor in the spinal cord may result inassociation of the NMDA receptor with PSD-95/SAP90 protein in spinalhyperalgesia.

Example 4

This example describes the experimental procedures used in theexperiments described in the examples 1-3.

Animal preparation and behavioral testing. All experiments were carriedout with the approval of the Animal Care Committee at the Johns HopkinsUniversity and were consistent with the ethical guidelines of theNational Institutes of Health and the International Association for theStudy of Pain. Male Sprague-Dawley rats (250-300 g) were implanted withan intrathecal PE-10 catheter into the subarachnoid space at the rostrallevel of the spinal cord lumbar enlargement through an incision at theatlanto-occipital membrane according to the method as described.^(25,27)One week or more later, the rats were injected intrathecally with salineor ONDs every 24 h for 4 days. On the fifth day, saline, NMDA,MK-801+NMDA or DNQX+NMDA was given intrathecally. Nociception wasevaluated by the radiant heat tail-flick test. The doses and time pointof maximal effect of NMDA used in the present study were determinedbased on a previous study.²⁴ The tail-flick apparatus (Model 33B TailFlick Analgesy Meter, IITC Life Science, Woodland Hills, Calif., USA)generated a beam of radiant heat that was focused on the underside ofthe tail, 5 cm from the tip. A cut-off time latency of 13.5 s was usedto avoid tissue damage to the tail. Nociception was assessed by the timerequired to induce tail-flick after applying radiant heat to the skin ofthe tail. The latency of reflexive removal of the tail from the heat wasmeasured automatically to the nearest 0.01 s. Tail-flick latency wasmeasured five times, and the basal latency was defined as the mean.Tail-flick data were expressed as percentage change calculated by theformula: (trial latency−baseline latency)/(baseline latency)×100%.Finally, PE-10 catheter position from each animal was confirmed whenlumbar enlargement segments were removed for western blot analysis.

PCR analysis of PSD-95/SAP90 in rat spinal cord. The cDNA sequencesencoding portions of the PSD-95/SAP90 were amplified using the followingsynthetic OND primers: PSD1 (5′-CAAGCCCAGCAATGCCTA-3′; SEQ ID NO: 3) andPSD2 (5′-CTTGTCGTAATCAAACAG-3′; SEQ ID NO: 4) for amplification ofPSD-95/SAP90 codon positions 789-1525. RNA samples (1 μg) from ratspinal cord, brain and muscle were reverse transcribed to generatefirst-strand cDNA. The PCR reactions were performed for 25 cycles. Eachcycle included 30 s at 94° C., 30 s at 55° C., and 30 s at 71° C. ThePCR products were directly cloned into the TA cloning vector (InvitrogenCo., San Diego, Calif., USA) and verified by automatic DNA sequencing.Fusion protein construction and preparation. cDNA sequence encodingportion of PSD-95/SAP90 was amplified by PCR and subcloned in-frame intoPGEX-2T (GIBCO, Rockville, Md., USA) via the BamHI and EcoRI restrictiondigest sites. The construct was then transformed into BL21 bacteria, andfollowing an induction of expression withisopropyl-β-D-thiogalactopyranoside, the protein was purified underdenaturing conditions using glutathione-coupled agarose. The aboveprotein was analyzed by SDS-PAGE followed by coomassie blue staining.

Isolation of PSD fraction. PSD fraction was prepared according toprocedures described by Luo et al¹¹ with modifications. In brief, thespinal cord and brain from male Sprague-Dawley rats were homogenized andcentrifuged at 800×g for 10 min to recover the supernatant S1 and thepellet P1. The S1 fraction was subjected to centrifugation at 7,100×gfor 15 min to obtain the pellet P2 and the supernatant S2. P2 wasresuspended and again subjected to centrifugation at 8,200×g for 15 minto recover the synaptosomal fraction P2′. The P2′ fraction was treatedwith an osmotic shock by diluting with double-distilled water andfurther centrifuged at 25,000×g for 20 min to generate the pellet LP1and the supernatant LS1. LP1 was resuspended and centrifuged at 33,000×gfor 20 min. The pellet LP1P was resuspended and loaded onto adiscontinuous sucrose gradient composed of 0.10, 1.5 and 2.0 M sucrose.After ultracentrifugation at 208,000×g for 2 h, the PSD fraction wasrecovered at the interface between 0.5 and 2.0 M sucrose. The PSDfraction was finally resuspended and centrifuged at 208,000×g for 30min. The recovered the pellet, resuspended in buffer, was considered asthe purified PSD fraction. Co-immunoprecipitation and immunoblotting.About 2-4 μg of the affinity-purified mouse PSD-95/SAP90 antibody(Upstate Biotechnology, Lake Placid, N.Y., USA) was preincubated with100 μl of a 1:1 slurry of protein A-sepharose for 1 h, and theprotein-antibody complex was spun down at 2,000 rpm for 4 min. Thesolubilized PSD fraction (400 μg) was then added to the sepharose beadsand the mixture incubated for 2-3 h at 4° C. The mixture was washed oncewith 1% TritonX-100 in immunoprecipitation buffer (137 mM NaCl, 2.7 mMKCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, 5 mM EGTA, 1 mM sodium vanadate, 10mM sodium pyrophosphate, 50 mM NaF, 20 U/ml Trasylol, and 0.1 mMphenylmethylsulfonyl fluoride), twice with 1% TritonX-100 inimmunoprecipitation buffer plus 300 mM NaCl, and three times withimmunoprecipitation buffer. The proteins were separated by SDS-PAGE andtransferred to a polyvinylidene difluoride membrane. In the controlgroups, PSD-95/SAP90 antibody was substituted with normal mouse serum,or was preincubated with excess of PSD-95/SAP90 fusion protein (100μg/ml). Immunoblotting was carried out as described by Lau et al¹⁰.Individual proteins were detected with the use of primary antibodies toPSD-95/SAP90 (1:1000), NMDA receptor subunits 2A/2B (1:200, ChemiconInternational Inc, Temecula, Calif., USA), nNOS (1:2000, Santa CruzBiotechnology Inc., Santa Cruz, Calif., USA), eNOS (1:500, TransductionLab., Lexington, Ky., USA) and SAP102 (gift from Dr. R. L. Huganir).Immunocytochemistry. Rats were perfused with 4% paraformaldehyde in 0.1M phosphate-buffered saline (PBS). The spinal cord was harvested andpostfixed at 4° C. for 4 h, and cryoprotected in 30% sucrose overnight.Sections (30 μm) were cut on a cryostat and then blocked for 1 h in PBScontaining 10% goat serum and 0.3% TritonX-100. Primary antibody toPSD-95/SAP90 (1:1000) was diluted into blocking reagent and incubatedwith sections overnight. Immunoperoxidase histochemistry was performedusing the ABC method. Control sections lacking primary antiserum werestained in parallel.

Example 5

This example demonstrates the decrease in threshold for isofluranecaused by inhibition of expression of PSD 95.

The value for isoflurane MAC in the control (saline-treated) group was1.16±0.08, which is consistent with that in the previousstudies.^(49,58) In the groups treated with the antisense ODNs at thedoses of 12.5, 25 and 50 μg, the isoflurane MACs were decreased fromisoflurane control MAC of 1%, 18% (P<0.01) and 44% (P<0.01),respectively (Table 1). In contrast, intrathecal administration of senseODN at the dose of 50 μg or missense ODN at the dose of 50 μg did notsignificantly change the value for isoflurane MAC compared to thecontrol group (Table 1).

No untoward effects were observed in any of the treated animalsincluding the antisense groups. In the ODN-treated groups, there was nosignificant change in either blood pressure or heart rate compared tocontrol group before the tail clamp (Table 2). Control baseline bloodpressure was 119.86±10.58 mmHg systolic and 106.36±7.78 mmHg diastolic,and control baseline heart rate was 513.00±40.28 beats/min.

Example 6

This example demonstrates that antisense ODN reduces the threshold forisoflurane, even in the presence of NMDA which increases the thresholdfor isoflurane.

In the saline-treated group, intrathecal NMDA at a dose of 1.25 μgcaused an increase from isoflurane control MAC by 15% (P<0.01; FIG. 1).The NMDA-induced change in isoflurane MAC was accompanied by asignificant increase in systolic and diastolic blood pressures(135.70±3.38 mmHg and 118.30±7.81 mmHg, respectively. P<0.05 vs control)but not in heart rate (529.20±55.20 beats/min, P>0.05 vs control).However, in the group pretreated with 50 μg of antisense ODN,intrathecal administration of 1.25 μg of NMDA did not result in asignificant increase in isoflurane MAC compared to the group treatedwith 50 μg of antisense ODN alone (P>0.05, FIG. 1). Interestingly, inthe group pretreated with 50 μg antisense ODN, intrathecal NMDA at adose of 1.25 μg still produced a significant increase in systolic anddiastolic blood pressures (138.00±5.77 mmHg and 117.00±6.35 mmHg,respectively. P<0.05 vs 50 μg antisense ODN-treated group alone) but notin heart rate (553±17 beats/min, P>0.05 vs control).

Example 7

This example demonstrates that antisense ODN did not affect locomotorfunction.

As shown in Table 3, ODNs with or without NMDA at the doses used in thepresent study did not produce significant effects on locomotor function.Convulsions and hypermobility were not observed in any of the treatedanimals including antisense ODN groups. In addition, there was nosignificant difference in general behaviors including spontaneousactivity between the control and the ODN-treated groups.

Example 8

This example demonstrates the materials and methods used in examples5-7.

The present study protocol was approved by the Animal Care Committee atthe Johns Hopkins University. Male Sprague-Dawley rats (250-300 g) werehoused individually in cages on a standard 12 h-12 h light-dark cycle.Water and food were available ad libitum until rats were transported tothe laboratory approximately 1 h before the experiments. All experimentswere performed under the same conditions.

Animal Preparation

Rats were anesthetized by intraperitoneal injection of pentobarbitalsodium (45 mg/kg). Chronic intrathecal catheters were inserted bypassing a polyethylene-10 (PE-10) catheter through an incision in theatlanto-occipital membrane to a position 8 cm caudal to the cisterna atthe level of the lumbar subarachnoid space using the methods describedpreviously.²⁷ The animals were allowed to recover for 5-7 days beforeexperiments were initiated. Rats that showed neurological deficitspostoperatively were removed from the study.

To examine whether the deficiency of the expression of PSD-95/SAP90affected the threshold for isoflurane anesthesia, we made an antisenseoligodeoxyribonucleotide (ODN) corresponding to the PSD-95/DLG/ZO-1(PDZ) domain nucleotides 241 to 258 (5′-TGTGATCTCCTCATACTC-3′; SEQ IDNO: 1) of rat PSD95/SAP90 mRNA, as well as the sense ODN and missenseODN (5′-AAGCCCTTGTTCCCATTT-3′; SEQ ID NO: 2).⁴⁹ All of the ODNs weresearched to exclude non-specificity of the sense or antisense ODNs andto show that missense ODN did not match any confounding sequences in theGenBank database (GenBank accession number M96853). The ODNs weredissolved in saline before administration. As described in the previouswork,⁴⁹ the rats were injected intrathecally with saline (10 μl)(control), antisense ODNs (12.5, 25, 50 μg/10 μl), sense ODN (50 μg/10μl) and missense ODN (50 μg/10 μl), respectively, followed by aninjection of 10 μl of saline to flush the catheter, every 24 h for 4days.

Measurement of MAC

On the fifth day after saline or ODNs injection, each rat was placed ina clear plastic cone and anesthetized with 5% isoflurane in oxygen forthree to five minutes. After tracheotomy, the trachea of each animal wasintubated with a 16-gauge polyethylene catheter. The inspired isofluraneconcentration was reduced to 2%, and the animals breathed spontaneouslyuntil cannulation of a carotid artery and a jugular vein with PE-50tubing was accomplished. The isoflurane concentration was decreasedfurther to 1.5%, and ventilation was controlled by a Harvard AnimalRespirator (Harvard Apparatus, South Natick, Mass.) adjusted accordingto the measurement of arterial blood gases to maintain normal partialpressure of oxygen (PO₂=91-94 mmHg), partial pressure of carbon dioxide(P_(CO2)=33−41 mmHg) and pH (7.4-7.44). Electrocardiography and systolicand diastolic blood pressure were monitored using a Grass Polygraph(Astroumed Grass, Quincy, Mass.) and Gould Pressure Transducer (Gould,Cleveland, Ohio). Rectal temperature was maintained between 36.5 and37.5° C. by use of a heating blanket and warming lights.

A PE-10 catheter was introduced through and beyond the endrotrachealtube until obstruction to passage was met and then withdrawn 1 to 2 mm.For isoflurane MAC measurement, the PE-10 catheter was connected to aparameter airway gas monitor (Datex-Engstrom, Inc., Tewksbury, Mass.).After stabilizing about 30 minutes, MAC was measured according to themethods described previously,³⁴ using a long hemostat (8-inch RochesterDean Hemostatic Forceps) clamped to the first ratchet lock on the tailfor 1 min. The tail was always stimulated proximal to a previous testsite. Gross movement of the head, extremities, or body was taken as apositive test result, whereas grimacing, swallowing, chewing, or tailflick were considered negative results. The isoflurane concentration wasreduced in decrements of 0.12 to 0.15% until the negative responsebecame positive, with 12-15 min equilibration allowed after changes inconcentration.^(50,51) The MAC was considered to be the concentrationmidway between the highest concentration that permitted movement inresponse to the stimulus and the lowest concentration that preventedmovement. Finally, intrathecal PE-10 catheter position from each animalwas confirmed.

In some saline-treated rats, after initial baseline MAC determination,NMDA at the dose of 1.25 μg³⁸ or saline was injected intrathecally in avolume of 10 μl saline, followed by an injection of 10 μl saline toflush the catheter. Fresh NMDA solution was prepared for eachexperiment. An isoflurane concentration was chosen at which movement didnot occur in the last negative response before the positive testresponse. At this isoflurane concentration, 10 min after the intrathecalinjection of NMDA, the animals were tested again for reactivity to tailclamp. The concentration of isoflurane was increased, and response totail clamp was checked every 12-15 min thereafter until a negativeresponse was achieved. In some antisense ODN (50 μg)-treated rats, afterinitial MAC determination, NMDA or saline was also administeredintrathecally. The MAC for isoflurane was again determined following theaforementioned procedures.

Tests of Locomotor Function

The effects of ODNs on locomotor function were examined using thefollowing methods.⁵² The animals were organized randomly into sixgroups: control (saline); 12.5 μg antisense ODN; 25 μg antisense ODN; 50μg antisense ODN; 50 μg sense ODN; 50 μg missense ODN. The rats werepretreated with ODNs or saline in the manner described above. On thefifth day, 10 μl of saline was injected intrathecally for each rat. Insome saline or antisense ODN (50 μg)-treated rats, fresh NMDA solution(1.25 μg/10 μl) was injected intrathecally. The following tests wereperformed with the experimenter blind to which group was treated withthe agents: (1) Placing reflex: The rat was held with the hind limbsslightly lower than the forelimbs, and the dorsal surfaces of the hindpaws were brought into contact with the edge of a table. Theexperimenter recorded whether the hind paws were placed on the tablesurface reflexively; (2) Grasping reflex: The rat was placed on a wiregrid and the experimenter recorded whether the hind paws grasped thewire on contact; (3) Righting reflex: The rat was placed on its back ona flat surface and the experimenter noted whether it immediately assumedthe normal upright position. Scores for placing, grasping and rightingreflexes were based on counts of each normal reflex exhibited in fivetrials. In addition, the rat general behaviors including spontaneousactivity were observed.

Statistical Analysis

The MAC data were assessed statistically by an analysis of variance.Intergroup differences were analyzed using the Newman-Keuls test.Locomotor data were assessed by a rank sum test. All data are reportedas the mean±SD. Significance was set at P<0.05.

Example 9

This example demonstrates the role of PSD95 in formalin-induced pain,which is a model for inflammatory-induced pain.

Pretreatment with PSD-95 antisense ODN produced significant decreases informalin-induced pain behaviors and c-fos expression in the spinal cord.Intrathecal antisense ODN at 50 μg reduced the number of flinches andshakes evoked by formalin by 59% (p<0.01) in the tonic period but not inthe phasic period. At the same dose, the antisense ODN also decreasedthe number of Fos-like immunoreactive neurons per section by 48%(p<0.05). However, the antisense ODN at 12.5 and 25 μg failed to producesignificant changes in the number of flinches and shakes in the phasicand tonic periods, or in the number of Fos-like immunoreactive neurons,when compared to the saline-treated group. Similarly, the sense ODN- andthe missense ODN-treated groups did not show any significant differencein the number of flinches and shakes in either period, when compared tothe saline-treated group.

These results demonstrate that PSD-95 antisense significantly reducedformalin-induced nociceptive behaviors in the tonic period but not inthe phasic period. This suggests that PSD-95 protein may play a key rolein the spinal sensitization induced by subcutaneous formalin injection.

All of the experiments were carried out with the approval of the AnimalCare Committee at the Johns Hopkins University. Thirty-eight maleSprague-Dawley rats (250-300 g, Hilltop Laboratory Animals, Scottsdale,Pa., USA) were implanted with an intrathecal PE-10 catheter at therostral level of the spinal cord lumbar enlargement according to themethod described by Yaksh and Rudy. After 4 to 7 days of recovery, theywere intrathecally injected with one of the following agents every 24hours for 4 consecutive days: saline (10 μl, n=6), PSD-95 antisenseoligodeoxynucleotide (ODN) (12.5 μg/10 μl, n=6; 25 μg/10 μl, n=6; 50μg/10 μl, n=6), sense ODN (50 μg/10 μl, n=5) or missense ODN (50 μg/10μl, n=9). On the fifth day, formalin (4%, 100 μl) was injected into oneof the hindpaws. The number of flinches and shakes of the injected pawwas assessed for 1 hour. The observational session was divided intophasic (0-10 min) and tonic (10-60 min) periods. Rats were sacrificedtwo hours after formalin injection and their lumbar spinal cords wereharvested for c-fos immunohistochemistry.

Data were assessed as mean±SD. Behavioral test and immunohistochemistryresults were assessed by ANOVA. Post-hoc testing was conducted usingBonferroni test. Significance was set at p<0.05.

Example 10

This example demonstrates that halothane inhibits the NMDA receptorsignaling pathway by inhibiting PDZ domain interactions between PSD-95or PSD-93 and NMDA receptors or nNOS.

Under normal conditions, PSD-95 interacts with nNOS, resulting in goodgrowth of the yeast carrying pGAD424-PSD-95 and pGBT9-nNOS in -LTHmedium. We found that halothane dose-dependently inhibited the growth ofthe yeast in -LTH media. Treatment with low halothane concentrations(0.4%-0.7%) slowed the growth of yeast clones. At high concentration(1.3%), halothane completely inhibited yeast growth. A similarphenomenon was observed in the growth of the yeast carryingpGAD424-PSD-95 and pGBT9-2B. The growth of the yeast carryingpGAD424-PSD-93 and pGBT9-nNOS or 2B was also inhibited by halothane in asimilar way. However, when these yeast clones grew in -LT instead of-LTH media in the presence of high halothane concentration (3.6%),growth did not differ from yeast grown without halothane. This findingsuggests that halothane is not cytotoxic to yeast. Rather, the failureof yeast to grow in -LTH media in the presence of halothane must be dueto disruption of protein-protein interactions by halothane. In addition,we used a biochemistry approach to demonstrate that halothane blocksGST-fusion PSD-95 or PSD-93 protein from binding to rat brain NMDAreceptors or to nNOS. These findings confirm the yeast two-hybridresults.

We utilized the yeast two-hybrid system to investigate the effects ofhalothane on protein interactions within the NMDA receptor signalingcomplex. The PDZ domain of nNOS or the C-terminus of NMDA receptorsubunit 2B (NR2B) was fused in frame with the GALA DNA-binding domain ina yeast vector, pGBT9. The PDZ domain of PSD95 or PSD-93 was fused inframe with the GAL4 activation domain in another yeast vector, pGAD424.Both yeast vectors were co-transformed into the Y190 yeast strain, whichwas then grown in the absence or presence of halothane at clinicallyrelevant concentrations. Protein-protein interactions were confirmed byboth yeast growth on -Leu/-Trp/-His (-LTH) medium and lacz expression.To confirm the yeast two-hybrid results, the GST fusion protein bindingassay was performed. The GST-fusion proteins, consisting of the secondPDZ domain of PSD-95 or PSD-93, were expressed in bacterial BL21 cellsand purified using glutathione-coupled agarose. After preincubation withor without different concentrations of balothane, GST-PSD-95 orGST-PSD-93 was incubated with membrane proteins from rat hippocampus atroom temperature for 1 h. After extensive washing, the bound proteinswere eluted by boiling in 1×SDS-PAGE sample buffer and detected byimmunoblotting.

Utilizing both the yeast two-hybrid system and protein binding assays,we found that halothane dose-dependently inhibited protein interactionsof PSD-95/NMDA receptor, PSD-95/nNOS, PSD-93/NMDA receptor andPSD-93/nNOS at physiological concentration. These proteininterconnections within the NMDA receptor signaling complex are believedto be critical for excitatory synaptic signal transduction. Disruptionof the signal complex may shed light on a novel mechanism for generalanesthesia.

Example 11

This example demonstrates the interaction of PSD-95/SAP90 with NMDAreceptor and neuronal nitric oxide synthase (nNOS) were examined.

We probed RNA from tissues of the spinal cord, other regions of brain(as positive control) and muscle (as negative control) for messagesencoding the PSD-95/SAP90 protein with the use of RT-PCR analysis. A0.735 Kb mRNA was detected in the spinal cord and the regions of brainbut not in muscle. The PCR product was directly cloned into the TAcloning vector and verified as PSD-95/SAP90 by automatic DNA sequencing.PSD-95/SAP90 protein also was found to enrich in the postsynapticdensity fraction of the spinal cord. Moreover, immunohistochemistryshowed that PSD-95/SAP90 was distributed mainly in spinal superficiallaminae, where PSD-95/SAP90 overlapped with NMDA receptor subunits 2A/2B(NR2A/2B) and nNOS, suggesting that PSD-95/SAP90 might interact withNR2A/2B and nNOS in the spinal cord. This was confirmed with the use ofco-immunoprecipitation, demonstrating that the PSD-95/SAP90 antibody wasable to immunoprecipitate not only PSD-95/SAP90 but also NR2A/2B andnNOS in vivo. In contrast, endothelial NOS was not immunoprecipitatedwith PSD-95/SAP90 antibody. The area-specific expression of PSD-95/SAP90and its interaction with NMDA receptor and nNOS in the spinal cord inthe present study suggest PSD-95/SAP90 may have important implicationsfor the mechanisms of nociceptive processing.

Example 12

This examples demonstrates the role of PSD-95/SAP90 in chronicneuropathic pain.

The effect of the deficiency of PSD-95/SAP90 on mechanical and thermalhyperalgesia in a rat neuropathic pain model was observed. The antisenseoligonucleotide (OND) specifically against PSD-95/SAP90 was employed toreduce the expression of PSD-95/SAP90 in spinal cord. The rats wereinjected intrathecally with saline (10 μl), antisense OND (50 μg/10 μl)or sense OND (50 μg/10 μl) every 24 h for 4 days. The unilateral L5spinal nerve was ligated. Hind paw withdrawal response to mechanical orheat stimuli was conducted 1 day prior to the surgery and at 3, 5, 7 and9 days postoperatively. In the saline-treated group, mechanical andthermal hyperalgesia developed within 3 days and persisted for 9 days orlonger. The pretreatment of antisense but not sense ODN resulted in asignificant delay of the onset of the mechanical and thermalhyperalgesia. Our results indicate that the deficiency of PSD-95/SAP90delayed the development of the neuropathic pain. PSD-95/SAP90 is likelyinvolved in the molecular mechanism of the production of hyperalgesia inneuropathic pain triggered via NMDA receptor activation.

REFERENCES

-   1. Aanonsen L. M., Lei S., and Wilcox G. L. (1990) Excitatory amino    acid receptors and nociceptive neurotransmission in rat spinal cord.    Pain 41, 309-321.-   2. Brenman J. E., Christopherson K. S., Craven S. E., McGee A. W.    and Bredt D. S. (1996) Cloning and characterization of postsynaptic    density 93, a nitric oxide synthase interacting protein. J.    Neurosci. 16, 7407-7415.-   3. Brenman J. E., Chao D. S., Gee S. H., McGee A. W., Craven S. E.,    Santillano D. R., Wu Z., Huang F., Xia H., Peters M. F.,    Froehner S. C. and Bredt D. S. (1996) Interaction of nitric oxide    synthase with the postsynaptic density protein PSD-95 and    α1-syntrophin mediated by PDZ domains. Cell 84, 757-767.-   4. Cho K. O., Hunt C. A. and Kennedy M. B. (1992) The rat brain    postsynaptic density fraction contains a homology of the drosophila    discs-large tumor suppressor protein. Neuron 9, 929-942.-   5. Christopherson K. S., Hillier B. J., Lim W. A. and    Bredt D. S. (1999) PSD-95 assembles a ternary complex with the    N-methyl-D-aspartic acid receptor and a bivalent neuronal NO    synthase PDZ domain. J. Biol. Chem. 274, 27467-27473.-   6. Kim E., Cho K. O., Rothschild A. and Sheng M. (1996)    Heteromultimerization and NMDA receptor-clustering activity of    chapsyn-110, a member of the PSD-95 family of proteins. Neuron 17,    103-113.-   7. Kistner U., Wenzel B. M., Veh R. W., Cases-Langhoff C., Garner A.    M., Appeltauer U., Voss B., Gundelfinger E. D. and    Garner C. C. (1993) SAP90, a rat presynaptic protein related to the    product of the drosophila tumor suppressor gene dig-A. J. Biol.    Chem. 268, 4580-4583.-   8. Kolhekar R., Meller S. T. and Gebhart G. F. (1993)    Characterization of the role of spinal N-methyl-D-aspartate    receptors in thermal nociception in the rat. Neuroscience 57,    385-395.-   9. Kornau H. C., Schenker L. T., Kennedy M. B. and    Seeburg P. H. (1995) Domain interaction between MDA receptor    subunits and the postsynaptic density protein PSD-95. Science 269,    1737-1740.-   10. Lau L.-H., Mammen A., Ehlers M. D., Kindler S., Chung W. J.,    Garner C. C. and Huganir R. L. (1996) Interaction of the    N-methyl-D-aspartate receptor complex with a novel    synapse-associated protein, SAP-102. J. Biol. Chem. 271,    21622-21628.-   11. Luo J., Wang Y., Yasuda R. P., Dunah A. W. and    Wolfe B. W. (1997) The majority of N-methyl-D-aspartate receptor    complexes in adult rat cerebral cortex contain at least three    different subunits. Mol. Pharmacol. 51, 79-86.-   12. Marvizon J. C., Martinez V., Grady E. F., Bunnett N. W. and    Mayer E. A. (1997) Neurokinin 1 receptor internalization in spinal    cord slices induced by dorsal root stimulation is mediated by NMDA    receptors. J. Neurosci. 17, 8129-36-   13. Meller S. T., Dykstra C. and Gebhart G. F. (1992) Production of    endogenous nitric oxide and activation of soluble guanylate cyclase    are required for N-methyl-D-aspartate-produced facilitation of the    nociceptive tail-flick reflex. Eur. J. Pharmacol. 214, 93-96.-   14. Meller S. T., Dykstra C. and Gebhart G. F. (1996) Acute thermal    hyperalgesia in the rat is produced by activation of    N-methyl-D-aspartate receptors and protein kinase C and production    of nitric oxide. Neuroscience 71, 327-335.-   15. Meller S. T. and Gebhart G. F. (1993) Nitric oxide (NO) and    nociceptive processing in the spinal cord. Pain 52, 127-136.-   16. Migaud M., Charlesworth P., Dempster M., Webster L. C.,    Watabe A. W., Makhinson M., He Y., Ramsay M. F., Morris R. G.,    Morrison J. H., O'Dell T. J. and Grant S. G. (1998) Enhanced    long-term potentiation and impaired learning in mice with mutant    postsynaptic density-95 protein. Nature 396, 433-439.-   17. Muller B. M., Kistner U., Kindler S., Chung W. J., Kuhlendahl    S., Fenster S. D., Lau L. F., Veh R. W., Huganir R. L.,    Gundelfinger E. D. and Garner C. C. (1996) SAP102, a novel    postsynaptic protein that interacts with NMDA receptor complexes in    vivo. Neuron 17, 255-265.-   18. Myers K. J. and Dean N. M. (2000) Sensible use of antisense: how    to use oligonucleotides as research tools. TiPS 21, 19-23.-   19. Nakanishi S. (1992) Molecular diversity of glutamate receptors    and implications for brain function. Science 258, 597-603.-   20. Niethammer M., Kim E. and Sheng M. (1996) Interaction between    the C terminus of NMDA receptor subunits and multiple membranes of    the PSD-95 family of membrane-associated guanylate kinase. J.    Neurosci. 16, 2157-2163.-   21. Randic M., Jiang M. C. and Cerne R. (1993) Long-term    potentiation and long-term depression of primary afferent    neurotransmission in the spinal cord. J. Neurosci. 13, 5228-5241.-   22. Rustioni A. and Weinberg R. J. (1989) The somatosensory system.    In: Handbook of Chemical Neuroanatomy (Bjorklund A, Hokfelt T,    Swanson L W, eds), pp 219-321. Amsterdam: Elsevier.-   23. Sattler R., Xiong Z., Lu W.-Y., Hafner M., MacDonald J. F. and    Tymianski M. (1999) Specific coupling of NMDA receptor activation to    nitric oxide neurotoxicity by PSD-95 protein. Science 284,    1845-1848.-   24. Siegan J. B. and Sagen J. (1995) Attenuation of NMDA-induced    spinal hypersensitivity by adrenal medullary transplants. Brain Res.    680, 88-98.-   25. Tao Y.-X., Hassan A., Haddad E. and Johns R. A. (2000)    Expression and action of cyclic GMP-dependent protein kinase Iα in    inflammatory hyperalgesia in rat spinal cord. Neuroscience 95,    525-533.-   26. Woolf C. J. and Thompson S. W. N. (1991) The induction and    maintenance of central sensitization is dependent on    N-methyl-D-aspartic acid receptor activation: implications for the    treatment of post-injury pain hypersensitivity states. Pain 44,    293-299.-   27. Yaksh T. L. and Rudy T. A. (1976) Analgesia mediated by a direct    spinal action of narcotics. Science 192, 1357-1358.-   28. Yung K. K. (1998) Localization of glutamate receptors in dorsal    horn of rat spinal cord. Neuroreport 9, 1639-1644-   29. Aanonsen L M, Wilcox G L: Nociceptive action of excitatory amino    acids in the mouse: effects of spinally administered opioids,    phencyclidine and sigma agonists. J Pharmacol Exp Ther 1987; 243:    9-19.-   30. Dickenson A H, Aydar E: Antagonism at the glycine site on the    NMDA receptor reduces spinal nociception in the rat. Neurosci Lett    1991; 121: 263-266.-   31. Kuroda Y, Strebel S, Rafferty C, Bullock R: Neuroprotective    doses of N-Methyl-D-aspartate receptor antagonists profoundly reduce    the minimum alveolar anesthetic concentration (MAC) for isoflurane    in rats. Anesth Analg 1993; 77: 795-800.-   32. Ishizaki K, Yoon D M, Yoshida N, Yamazaki M, Arai K, Fujita T:    Intrathecal administration of N-Methyl-D-aspartate receptor    antagonist reduces the minimum alveolar anesthetic concentration of    isoflurane in rats. Br J Anaesth 1995; 75: 636-638.-   33. Ishizaki K, Yoshida N, Yoon D M, Yoon M H, Sudoh M, Fujita T:    Intrathecally administered NMDA receptor antagonists reduce the MAC    of isoflurane in rats. Can J Anaesth 1996; 43: 724-730.-   34. Davies S N, Lodge D: Evidence for involvement of    N-methyl-D-aspartate receptors in ‘wind-up’ of class 2 neurons in    the dorsal horn of the rat. Brain Res 1987; 424: 402-406.-   35. Dickenson A H, Sullivan A F: Evidence for a role of the NMDA    receptor in the frequency dependent potentiation of deep dorsal horn    neurons following C-fiber stimulation. Neuropharmacology 1987; 26:    1235-1238.-   36. Dougherty P M, Willis W D: Enhancement of spinalthalamic neuron    responses to chemical and mechanical stimuli following combined    micro-iontophoretic application of N-methyl-D-aspartic acid and    substance P. Pain 1991; 47: 85-93.-   37. Malmberg A B, Yaksh T L: Hyperalgesia mediated by spinal    glutamate or substance P receptor blocked by spinal cyclooxygenase    inhibition. Science 1992; 257: 1276-1279.-   38. Kawamata T, Omote K: Activation of spinal N-methyl-D-aspartate    receptors stimulates a nitric oxide/cyclic guanosine    3,5-monophosphate/glutamate release cascade in nociceptive    signaling. Anesthesiology 1999; 91: 1415-1424.-   39. Tao Y X, Johns R A: Activation of cGMP-dependent protein kinase    Iα is required for N-methyl-D-aspartate- or nitric oxide-produced    spinal thermal hyperalgesia. Eur J Pharmacol 2000; 392: 141-145.-   40. Davar G, Hama A, Deykin A, Vos B, Maciewicz R: MK-801 blocks the    development of thermal hyperalgesia in a rat model of experimental    painful neuropathy. Brain Res 1991; 553: 327-330.-   41. Mao J, Price D D, Mayer D J, Lu J, Hayes R L: Intrathecal MK-801    and local nerve anesthesia synergistically reduce nociceptive    behaviors in rats with peripheral mononeuropathy. Brain Res 1992;    576: 254-262.-   42. Ren K, Dubner R: NMDA receptor antagonists attenuate mechanical    hyperalgesia in rats with unilateral inflammation of the hindpaw.    Neurosci Lett 1993; 163: 22-26.-   43. Seltzer Z, Cohn S, Ginzburg R, Behavior in rats by spinal    disinhibition and NMDA receptor blockade of injury discharge. Pain    1991; 45: 69-75.-   44. Yamamoto T, Shimoyama N, Mizuguchi T: The effect of morphine,    MK-801, an NMDA antagonist, and CP-96,345, an NK-1 antagonist, on    the hyperalgesia evoked by carrageenan injection in the rat paw.    Anesthesiology 1993; 78: 124-133.-   45. Kennedy M B: The postsynaptic density at glutamatergic synapses.    Trends Neurosci 1997; 20: 264-268.-   46. Nagano T, Jourdi H, Nawa H: Emerging roles of Dlg-like PDZ    protein in the organization of the NMDA-type glutamatergic synapse.    J Biochem 1998; 124: 869-875.-   47. Hata Y, Nakanishi H, Takai Y: Synaptic PDZ domain-containing    proteins. Neurosci Res 1998; 32: 1-7.-   48. O'Brien R J, Lau L F, Huganir R L: Molecular mechanisms of    glutamate receptor clustering at excitatory synapses. Curr Opin    Neurobiol 1998; 8: 364-369.-   49. Tao Y X, Huang Y Z, Mei L, Johns R A: Expression of PSD-95/SAP90    is critical for N-methyl-D-aspartic acid receptor-mediated thermal    hyperalgesia in the spinal cord. Neuroscience 2000; 98: 201-206.-   50. Tao Y X, Hassan A, Johns R A: Intrathecally administered    cGMP-dependent protein kinase Iα inhibitor significantly reduced the    threshold for isoflurane anesthesia. Anesthesiology; 2000: 493-499.-   51. Eger E I, Saidman L J, Brandstater B: Minimum alveolar    anesthetic concentration: A standard of anesthetic potency.    Anesthesiology 1965; 26: 756-763.-   52. Eger E I: Effect of inspired anesthetic concentration on the    rate of alveolar concentration. Anesthesiology 1963; 26: 153-157.-   53. Coderre T J, Van Empel I: The utility of excitatory amino acid    (EAA) antagonist as analgesic agents. I. Comparison of the    antinociceptive activity of various classes of EAA antagonist in    mechanical, thermal and chemical nociceptive tests. Pain 1994; 59:    345-352.-   54. Pajewski T N, DiFazio C A, Moscicki J C, Johns R A: Nitric oxide    synthase inhibitor, 7-nitro indazole and nitroG-L-arginine methyl    ester, dose dependently reduce the threshold for isoflurane    anesthesia. Anesthesiology 1996; 85: 1111-1119.-   55. Hunt C A, Schenker L J, Kennedy M B: PSD-95 is associated with    the postsynaptic density and not with the presynaptic membrane at    forebrain synapses. J Neurosci 1996; 16: 1380-1388.-   56. Valtschanoff J G, Burette A, Wenthold R J, Weinberg R J:    Expression of NR2 receptor subunit in rat somatic sensory cortex:    synaptic distribution and colocalization with NR1 and PSD-95. J Comp    Neurol 1999; 410: 599-611.-   57. Garcia R A, Vasudevan K, Buonanno A: The neuregulin receptor    ErbB-4 interacts with PDZ-containing proteins at neuronal synapses.    Proc Natl Acad Sci USA 2000; 97: 3596-601.-   58. Coote J H: The organization of cardiovascular neurons in the    spinal cord. Rev Physiol Pharmacol 1988; 110: 147-285.-   59. Loewy A D, Spyer K M: Central Regulation of autonomic functions.    Oxford, UK: Oxford Uni Press, 1999.-   60. Morrison S F, Callaway T A, Milner T A, Reis D J: Glutamate in    the spinal sympathetic intermediolateral nucleus: location by light    and electron microscopy. Brain Res 1989; 503: 5-15.-   61. Morrison S F, Ernsberger P, Milner T A, Callaway T A, Gong A,    Reis D J: A glutamate mechanism in the internediolateral nucleus    mediates sympathoexcitatory responses to stimulation of the rostral    ventrolateral medulla. Prog Brain Res 1989; 81: 159-169.-   62. Hong Y, Henry J L: Glutamate, NMDA and NMDA receptor    antagonists: cardiovascular effects of intrathecal administration in    the rat. Brain Res 1992; 569: 38-45.-   63. West M, Huang W: Spinal cord excitatory amino acids and    cardiovascular autonomic responses. Am J Physiol 1994; 267:    H865-873.-   64. Hong Y, Yashpal K, Henry J L: Cardiovascular responses to    intrathecal administration of strychnine in the rat: Brain Res 1989;    169-173.

TABLE 1 Effects of the suppression of the expression of PSD-95/SAP90 inthe spinal cord on the N-methyl-D-aspartate-induced thermal hyperalgesiaMK-801 + DNQX + AS (25 μg) + AS (50 μg) + SE (50 μg) + MS (50 μg) +Control NMDA NMDA NMDA NMDA NMDA NMDA NMDA ΔTF latency (%) −1.23 ±−25.84 ± 0.9 ± −22.6 ± −11.65 ± −4.72 ± −21.48 ± −20.96 ± 1.48 1.91*3.0*** 3.13* 2.46**^(,)**** 2.49*** 1.55* 1.68* Percentage change of TFlatency was calculated as described in the Experimental Procedures. AS:antisense; SE: sence; MS: missense. Data are presented as mean ± S.E.M.of six to 12 animals in each group. *P < 0.01 significantly differentfrom control. **P < 0.05 significantly different from control. ***P <0.01 significantly different from NMDA alone. ****P < 0.05 significantlydifferent from NMDA alone.

TABLE 2 Effects of Antisense (AS), Sense (SE), and Missense (SE)Oligodeoxyribonucleotides and Saline on Isoflurane MAC, Blood Pressure(BP), and Heart Rate Saline 12.5 μg AS 25 μg AS 50 μg AS 50 μg SE 50 μgMS (n = 14) (n = 6) (n = 6) (n = 6) (n = 6) (n = 6) MAC  1.16 ± 0.08 1.15 ± 0.18  0.98 ± 0.14*  0.72 ± 0.05*  1.15 ± 0.21  1.13 ± 0.15 BP(mmHg) Systolic 119.86 ± 10.58 127.58 ± 11.72 122.75 ± 10.81 129.58 ±11.73 126.67 ± 10.40 121.33 ± 15.84 Diastolic 106.36 ± 7.78 112.58 ±7.14 105.83 ± 7.89 112.50 ± 11.20 105.58 ± 13.07 105.75 ± 11.40 Heartrate (beats/min) 513.00 ± 40.78 534.80 ± 29.13 541.20 ± 16.70 514.20 ±62.20 529.60 ± 22.61 524.70 ± 44.90 *P < 0.01 versus saline-treated(control) group. MAC = minimum alveolar concentration.

TABLE 3 Mean (SD) Changes in Locomotor Test Agents Placing GraspingRighting Saline 5 (0) 5 (0) 5 (0) 12.5 μg AS   5 (0) 5 (0) 5 (0) 25 μgAS 5 (0) 5 (0) 5 (0) 50 μg AS 4.83 (0.41) 4.67 (0.52) 4.83 (0.41) 50 μgSE 5 (0) 5 (0) 5 (0) 50 μg MS 5 (0) 5 (0) 5 (0) Saline + 5 (0) 5 (0) 5(0) 1.25 μg NMDA 50 μg AS + 4.83 (0.41) 4.83 (0.41) 4.83 (0.41) 1.25 μgNMDA N = 6, five trials. AS = antisense; SE = sense; MS = missense; NMDA= N-methyl-o-aspartate.

1. A method of screening for substances useful for relieving pain orinducing unconsciousness or sedation, comprising: contacting a testsubstance with a first protein and a second protein under conditionswhere the first protein and the second protein bind to each other,wherein the first protein is selected from the group consisting ofPSD93, PSD95, and a combination thereof, wherein the second protein isselected from the group consisting of nNOS, NMDA receptor, NR2A subunit,NR2B subunit, and combinations thereof; determining an amount selectedfrom the group consisting of: free nNOS, free PSD93, free PSD95, freeNMDA receptor, free NR2A subunit, free NR2B subunit, bound nNOS, boundPSD93, bound PSD95, bound NMDA receptor, bound NR2A subunit, bound NR2Bsubunit and combinations thereof; identifying a test substance whichincreases the amount of free nNOS, free PSD93, free PSD95, free NMDAreceptor, free NR2A subunit, or free NR2B subunit, or which decreasesthe amount of bound nNOS, bound PSD93, bound PSD95, bound NMDA receptor,bound NR2A subunit, or bound NR2B subunit as a candidate drug forrelieving pain or inducing unconsciousness or sedation.
 2. The method ofclaim 1 wherein the step of contacting is done in vitro.
 3. The methodof claim 1 wherein the step of contacting is done in yeast cellscontaining recombinant forms of the first and second proteins.
 4. Themethod of claim 3 wherein the first and second recombinant proteins areeach fused to a first and second yeast protein, wherein the first andsecond yeast proteins reconstitute a functional transcriptionalactivator when brought into physical proximity by binding of the firstrecombinant protein to the second recombinant protein.
 5. The method ofclaim 1 further comprising the step of: testing an identified candidatedrug in an animal to determine if the candidate drug relieves pain orinduces unconsciousness or sedation.
 6. The method of claim 1 whereinthe test substance is contacted with PSD95 and nNOS.
 7. The method ofclaim 1 wherein the test substance is contacted with PSD95 and NMDAreceptor.
 8. The method of claim 1 wherein the test substance iscontacted with PSD95, nNOS, and NMDA receptor.
 9. The method of claim 1wherein the test substance is contacted with PSD95 and NR2A.
 10. Themethod of claim 1 wherein the test substance is contacted with PSD95 andNR2B.
 11. The method of claim 1 wherein the test substance is contactedwith PSD93 and nNOS.
 12. The method of claim 1 wherein the testsubstance is contacted with PSD93 and NMDA receptor.
 13. The method ofclaim 1 wherein the test substance is contacted with PSD93, nNOS, andNMDA receptor.
 14. The method of claim 1 wherein the test substance iscontacted with PSD93 and NR2A.
 15. The method of claim 1 wherein thetest substance is contacted with PSD93 and NR2B.
 16. The method of claim1 wherein surface plasmon resonance is used to determine said amount.17. The method of claim 1 wherein antibodies are used to determine saidamount.